The thymoproteasome subunit b5t is specifically expressed in cortical thymic epithelial cells (TECs) and generates unique peptides to support positive selection. In this study, using a mouse model ubiquitously expressing b5t, we showed that aberrant expression of self-peptides generated by b5t affects CD8 + T cell homeostasis, including thymic selection and maintenance of the peripheral naive pool of CD8 + T cells. In mice in which b5t was expressed both in cortical and medullary TECs, the abundance of CD8 + lineage thymocytes was reduced, and extra-thymic expression of b5t caused accumulation of CD8 + T cells with the memory or exhausted phenotype and induced autoreactive T cell responses. We found that thymoproteasomes are essential for positive selection but that the subsequent change in peptide repertoire in the medulla is also crucial for thymic selection and that b5t-derived peptide must be confined to the thymus to avoid autoimmunity in peripheral tissues.

To quantitatively categorize protein structures, we developed a quantitative coarse-grained model of protein structures with a novel amino acid network, the interaction selective network (ISN), characterized by the links based on interactions in both the main and side chains. We found that the ISN is a novel robust network model to show the higher classification probability in the plots of average vertex degree (k) versus average clustering coefficient (C), both of which are typical network parameters for protein structures, and successfully distinguished between “all-α” and “all-β” proteins. On the other hand, one of the typical conventional networks, the α-carbon network (CAN), was found to be less robust than the ISN, and another typical network, atomic distance network (ADN), failed to distinguish between these two protein structures. Considering that the links in the CAN and ADN are defined by the interactions only between the main chain atoms and by the distance of the closest atom pair between the two amino acid residues, respectively, we can conclude that reflecting structural information from both secondary and tertiary structures in the network parameters improves the quantitative evaluation and robustness in network models, resulting in a quantitative and more robust description of three-dimensional protein structures in the ISN.

To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (ΔV u ) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. ΔV u values for the unfolding to urea-and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56±5 and 29±1 mL mol -1 , respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive ΔV u reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt c than in urea-denatured Cyt c, resulting in the smaller positive ΔV u . On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted ΔV u (4±2 mL mol -1 ) in urea-denatured Cyt c. The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt c by estimating ΔV u , which allows us to discuss the hydrated structures in the denatured states of proteins.In aqueous solutions, linear protein polypeptide chains decrease in entropy and collapse into a globule to minimize the surface area that is exposed to the solvent. The folded state is a low-entropy subensemble in all possible collapsed globular conformations for the protein chain. In contrast, the unfolded state is an ensemble that is not or much less structured and has higher entropy than the folded state does. For folding to be beneficial, the folded state must be sufficiently and energetically favorable to overwhelm the higher entropy associated with structural disorder within the globular phase. The major energetic driving force originates from the van der Waals, hydrogen bonding, and electrostatic interactions, both within the polypeptide chain and between the chain andThe high pressure absorption spectroscopy revealed that the partial volume changes for the unfolding of cytochrome c (Cyt c) to the urea-and guanidine hydrochloride (GdnHCl)-denatured unfolded states (ΔV u ) were positive, reflecting hydration to hydrophobic heme; however, a more positively shifted ΔV u was observed for urea-denatured Cyt c. Introduction of the mutation near the axial ligand induced more drastic changes in the hydrated structure of the urea-denatured Cyt c, suggesting that the hydrated structure in the unfolded state depends on the denaturant. Our approach enables us to examine the dehydration associated with protein folding and hydration structures in the unfolded states.

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